Nobuyuki Minami

Predicting response to plasma exchange in patients with thrombotic thrombocytopenic purpura with measurement of vWF‐cleaving protease activity

BACKGROUND: Severe deficiency of vWF‐cleaving protease (vWF‐CPase) activity was recently found in patients with thrombotic thrombocytopenic purpura (TTP). Although the survival of patients with TTP has been dramatically improved with plasma exchange (PE), there are still many patients who are refractory to PE and immunosuppressive therapy.

Activity of interleukin 6 in the differentiation of monocytes to macrophages and dendritic cells

Peripheral blood monocytes are common precursor cells of dendritic cells (DCs) and macrophages. We have searched for factors with the potential to regulate the differentiation of monocytes to DCs and macrophages. When CD14+ monocytes are cultured with granulocyte–macrophage colony‐stimulating factor (GM‐CSF) and interleukin (IL) 4, the CD14+CD1a− population, which consists of macrophages, was found in the serum‐containing cultures but not in the serum‐free cultures. Addition of IL‐6 receptor‐neutralizing monoclonal antibody (mAb) or gp130‐neutralizing mAb to the serum‐containing cultures resulted in a decreased population of CD14+CD1a− cells. An increase in the CD14+CD1a− population with reduction in CD14−CD1a+ DCs was observed with the addition of IL‐6 to cultures, whereas IL‐11, leukaemia inhibitory factor, oncostatin M or macrophage colony‐stimulating factor did not affect the differentiation of monocytes in the presence of GM‐CSF plus IL‐4. This effect of IL‐6 was blocked by tumour necrosis factor α (TNF‐α), lipopolysaccharide (LPS), IL‐1β, CD40 ligand (CD40L) and transforming growth factor β1 (TGF‐β1). Among these factors, TNF‐α was most potent in interfering with the action of IL‐6. These results suggest that IL‐6 inhibits the differentiation of monocytes to DCs by promoting their differentiation toward macrophages, which is modulated by factors such as TNF‐α, LPS, IL‐1β, CD40L and TGF‐β1.

The Soluble Notch Ligand, Jagged-1, Inhibits Proliferation of CD34 + Macrophage Progenitors

The Notch/Notch ligand system controls diverse cellular processes. The proteolytic cleavage generates transmembrane and soluble forms of Notch ligands.We examined the effect of a soluble Notch ligand, human Jagged-1, on human cord blood (CB) CD34+ cells, under serum-deprived conditions, using soluble human Jagged-1—immunoglobulin G1 chimera protein (hJagged-1). Soluble hJagged-1 inhibited myeloid colony formation but not erythroid-mix or erythroid colony formation, in the presence of stem cell factor (SCF), interleukin-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, thrombopoietin, and erythropoietin. Cytological analysis revealed that the decrease in myeloid colonies resulted mainly from the inhibition of macrophage colony formation. Furthermore, soluble hJagged-1 led to the inhibition of macrophage colony formation supported by M-CSF plus SCF and GM-CSF plus SCF. Delayed-addition experiments and the analysis of colony sizes demonstrated that soluble hJagged-1 inhibited the growth of macrophage progenitors by acting in the early stage of macrophage development. The direct action of hJagged-1 was confirmed by the enhanced expression of the HES-1 (hairy enhancer of the split-1) gene. These results suggest that soluble hJagged-1 may regulate human hematopoiesis in the monocyte/macrophage lineage.

Efficient ex vivo generation of dendritic cells from CD14+ blood monocytes in the presence of human serum albumin for use in clinical vaccine trials

Dendritic cells (DC) with the potential to induce anti‐tumour immunity represent one of the promising candidates for cancer vaccines. Efficiency of ex vivo DC generation depends on culture conditions, especially protein components in the plasma or serum used. Using human serum albumin (HSA), we devised a constant and reproducible culture method for DC generation from peripheral blood CD14+ cells. The number of DC obtained with 2% HSA‐supplemented cultures containing granulocyte‐macrophage colony‐stimulating factor and interleukin 4 were consistently higher than in cultures with various concentrations of autologous plasma or serum. The concentrations and time points tested for plasma or serum considerably affected the number of DC recovered. DC prepared with HSA acquired the ability to uptake dextran, and expressed high levels of major histocompatibility (MHC) and co‐stimulatory molecules similar to DC cultured with autologous plasma or serum. Although DC cultured with autologous plasma or serum consisted of CD1a+ and CD1a− populations, DC differentiated in the presence of HSA expressed CD1a. DC obtained with HSA primed and induced immunogenic peptide‐specific cytotoxic T lymphocytes against a tumour rejection antigen, HER2. These findings suggest that our method for preparation of DC with HSA should prove valuable in DC generation for immunotherapy.

Activity of the ligand for c-mpl, thrombopoietin, in early haemopoiesis.

We examined the role of the ligand for c‐mpl, thrombopoietin (TPO), in murine early haemopoiesis, using a serum‐free culture system. TPO in combination with the ligand for c‐kit (SF) or interleukin‐3 (IL‐3) supported colony formation by marrow cells of 5‐fluorouracil (5‐FU)‐treated mice, whereas TPO alone yielded no colony. When blast cell colonies grown in the presence of TPO plus SF or TPO plus IL‐3 were individually replated in suspension cultures containing serum and several growth factors, various combinations of myeloid lineages were seen, indicating that the progenitors supported by TPO plus SF or TPO plus IL‐3 are multipotential. Delayed addition experiments demonstrated that TPO has the potential to effectively support the survival of haemopoietic progenitors. We then studied the effects of TPO on proliferative kinetics of cycling progenitors. TPO hastened IL‐3‐dependent growth of progenitors by shortening the time required for cell cycling. These results suggest that TPO, as a single factor, can support the survival of haemopoietic progenitors and TPO synergizes with SF or IL‐3 to act on early multipotential haemopoietic progenitors.

Possible involvement of bcl-2 in regulation of cell-cycle progression of haemopoietic cells by transforming growth factor-β1

Transforming growth factor‐β1 (TGF‐β1) acts directly on haemopoietic progenitor cells to regulate their growth. To investigate a possible link between the action of TGF‐β1 and cell death regulators such as bcl‐2, we utilized Ba/F3 cells, the interleukin‐3 (IL‐3)‐dependent growth of which could be modulated by TGF‐β1, as well as haemopoietic progenitor cells. We demonstrate here that up‐regulation of bcl‐2 protein (Bcl‐2) as well as that of an inhibitor of cyclin/cyclin‐dependent kinase complex, p27, was associated with TGF‐β1‐induced deceleration of the cell‐cycling of haemopoietic progenitor cells and Ba/F3 cells. The data from cell‐cycle analysis of Ba/F3 cells showed that TGF‐β1 retarded the G1 to S phase transition. Analysis of cells with the potential to express Bcl‐2 in an inducible manner indicated that up‐regulation of Bcl‐2 was sufficient for not only an increase in the level of p27 but also to inhibit the cell growth. Using c‐kit‐overexpressing cells, we observed that the potential of TGF‐β1 to up‐regulate the expression of Bcl‐2 and p27 could be counteracted by the c‐kit ligand, stem cell factor. These results demonstrate that Bcl‐2 exerts an essential function in the regulation of G1 to S phase transition of haemopoietic cells by TGF‐β1.

Increased Plasma Thrombomodulin as a Vascular Endothelial Cell Marker in Patients With Thrombotic Thrombocytopenic Purpura and Hemolytic Uremic Syndrome

Several hemostatic and vascular endothelial cell markers were measured in 39 patients with thrombotic thrombocytopenic purpura (TTP)/hemolytic uremic syndrome (HUS) and in 20 healthy volunteers to examine the relationship between the occurrence of hemostatic abnormality or vascular endothelial cell injury and patient outcome. The plasma levels of von Willebrand factor, tissue plasminogen activator (TPA), plasminogen activator inhibitor (PAI-1), and the TPA-PAI-1 complex were significantly increased in TTP/HUS patients; however, the levels of these markers were not significantly different between TTP/HUS patients who survived and those who died, suggesting that these markers might not be directly related to outcome. The plasma levels of soluble granule membrane protein (GMP)-140 were significantly higher in TTP/ HUS patients than in healthy volunteers, suggesting that platelets and vascular endothelial cells are activated or injured in TTP/HUS. There was no significant difference in GMP-140 levels between TTP/HUS patients with good and poor prognoses ; this may be owing to the release of GMP-140 from platelets. The plasma thrombomodulin (TM) levels in TTP/ HUS patients were significantly higher than in healthy volunteers; the plasma TM levels were significantly higher in patients who died than in patients who survived. These findings showed that TM levels reflect the outcome and that the outcome of TTP/HUS depends on the presence vascular endothelial cell injury. The plasma protein C and antithrombin levels were markedly reduced in TTP/HUS patients who died compared with those who survived. These findings suggest that reduced plasma antithrombin and protein C may be useful markers of systemic vascular endothelial injury. In conclusion, the results of this study showed that the outcome of TTP/HUS is related to vascular endothelial cell injury and that plasma TM, antithrombin, and protein C levels may be useful markers of systemic vascular endothelial cell injury.

Granulocyte colony-stimulating factor and its receptor in acute promyelocytic leukemia

Expression of granulocyte colony‐stimulating factor (G‐CSF) receptor (G‐CSFR) and in vitro proliferative response to G‐CSF were investigated by quantitative immunofluorescence and [3H] thymidine uptake, respectively, in a series of acute myeloid leukemias (AML). The results indicated that G‐CSFR was detected at high levels in acute promyelocytic leukemia (APL) cells, in comparison with other types of AML. Moreover, APL cells were also seen to predominantly proliferate in response to G‐CSF. Based on these observations, we administered recombinant human G‐CSF to a patient with APL in the third relapse that was resistant to both cytotoxic agents and all trans retinoic acid, in an attempt to sensitize the leukemic cells to cell‐cycle‐dependent agents. Complete remission was achieved. The finding that APL cells are exquisitely responsive to G‐CSF supports the view that G‐CSF is useful for augmentation of their vulnerability to cell‐cycle specific agents. Am. J. Hematol. 58:31–35, 1998.

Plasma sFas and sFas ligand levels in patients with thrombotic thrombocytopenic purpura and in those with disseminated intravascular coagulation

Fas, a member of the tumor necrosis receptor superfamily, is 36 kD surface protein containing a single transmembrane region and induces apoptosis by Fas–Fas ligand binding. Soluble Fas (sFas) is produced as the form lacking 21 amino acid residues containing the transmembrane domain by alternative splicing. We found that the plasma sFas levels of 33 patients with thrombotic thrombocytopenic purpura/hemolytic uremic syndrome (TTP/HUS), 19 patients with disseminated intravascular coagulation (DIC), and 10 non‐DIC patients with multiple organ failure (MOF) were significantly higher than those of 21 non‐DIC patients without organ failure and those of 25 healthy volunteers. The plasma sFas ligand levels of the TTP/HUS patients, the DIC patients, and the non‐DIC patients with MOF were significantly higher than those of the non‐DIC patients without organ failure and those of the healthy volunteers. The plasma sFas levels were significantly correlated with the plasma sFas ligand levels in all subjects. The plasma thrombomodulin (TM) levels were increased significantly in the TTP/HUS patients, the DIC patients, and the non‐DIC patients with MOF compared with the levels of the non‐DIC patients without organ failure and the healthy volunteers. The plasma sFas antigen levels were correlated significantly with the plasma TM levels in all subjects. These findings suggest that the increases of sFas and sFas ligand that cause apoptosis might be related to the vascular endothelial cell injuries in TTP and DIC with organ failure. Am. J. Hematol. 61:21–25, 1999.

Two Independent Clones in Myelodysplastic Syndrome Following Treatment of Acute Myeloid Leukemia

We describe a 55-year-old Japanese woman with therapy-related myelodysplastic syndrome (t-MDS) with 2 independent clones, t(1;2)(p36;p21) and t(11;12)(p15;q13). She was diagnosed with acute myeloid leukemia (AML) with cytological features of the bone marrow and peripheral blood. Cytogenetic evaluation revealed a 46,XX karyotype. She received chemotherapy and achieved complete remission (CR). Despite maintenance chemotherapy, she suffered a relapse. Chromosomal analysis showed t(1;2)(p36;p21) in 2 of 20 metaphases. At second CR, this clone transiently disappeared. Nine months later, t(1;2) (p36;p21) was detected again in 3 of 20 metaphases while the patient remained in CR. Six months later, bone marrow examination disclosed trilineage dysplasia without an excess of blasts, suggesting MDS. t(1;2)(p36;p21) was observed in 16 of 20 metaphases. The clinical course and serial cytogenetic findings were diagnostic of t-MDS. The duration of t-MDS was 6 years. During this period, persistent t(1;2)(p36;p21) and transient t(11;12)(p15;q13) were found.When t-MDS evolved to AML, cytogenetic evaluation revealed 46,XX,t(1;2)(p36;p21),del(7)(q22),add(19)(p13).